1. Field of the Invention (Technical Field)
The present invention relates to methods for identification and determination of target-specific sequences within peptides and proteins; methods to determine the specific sequence of that portion of peptides or proteins that bind to a receptor or target of interest, or mediate a biological activity of interest; and methods to determine the target-specific folding site within peptides and proteins.
2. Background Art
Note that the following discussion refers to a number of publications by author(s) and year of publication, and that due to recent publication dates certain publications are not to be considered as prior art vis-à-vis the present invention. Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes.
Peptide and Protein Folding. Determination of the biologically relevant structure of proteins and peptides, which can be characterized as a functional three-dimensional structure, is a difficult problem in the biological, biochemical and pharmaceutical sciences. Through use of any of a variety of methods the primary structure of relevant peptides or proteins may be ascertained. That is, the sequence of amino acid residues composing the peptide or protein can be determined, and it is known that the peptide or protein has a desired biological effect, such as binding a target molecule or receptor of interest, mediating a biological activity of interest, or the like. However, both the three-dimensional structure and identification of the specific portion of the peptide or protein forming a ligand and thereby giving rise to the desired biological effect is frequently unknown.
Peptides and proteins are highly flexible, due in large part to the high rotational degrees of freedom of individual amino acid residues. In addition, some bonds in side chains of individual amino acid residues also have rotational degrees of freedom. The non-bonded steric interactions between amino acid residues force the peptide or protein along its degrees of freedom into some stable minimal free energy configuration. Local structures, also known as a “secondary structure,” are common in peptides and proteins. These structures include α-helixes, β-bends, sheets, extended chains, loops and the like, and most often contribute to binding or receptor-specificity of peptides and proteins.
There are several types of α-helixes known, differing in torsion angles within the amino acid residues of the actual turn and by the patterns of intra- and inter-molecular hydrogen bonding. There are also a number of known different β-bends, differing in the dihedral torsion angles ψ (for the Ca—C bond) or φ (for the Ca—N bond), or both.
Peptide and protein folding are recognized as complex problems, involving consideration of all or virtually all the primary structure of the peptide or protein. Thus distal portions of a given molecule may significantly and substantially affect the secondary structure of a portion of the molecule of interest. For example, a six amino acid residue peptide may, as a distinct molecule, have a substantially different secondary and/or tertiary structure than would that same sequence as part of a larger peptide or protein.
A wide variety of mathematical, computational and others models have been developed for predicting the secondary structure of proteins and the secondary and tertiary structure of peptides, but no model gives satisfactory responses under other than the most limited circumstances. For example, software modeling programs (e.g., such as those distributed by Tripos, Inc., Pharmacopeia Inc. and the like), depend on various algorithms, statistical tools, assumed relationships between groups and the like, any or all of which may not be valid for any given protein or peptide. A number of methods are described in the art, such as those disclosed in International Publication No. WO 00/23564 to Xencor, Inc., International Publication Nos. WO 00/57309 and WO 01/35316, both to Structural Bioinformatics, Inc., International Publication No. WO 01/50355 to Structural Bioinformatics Advanced Technologies A/S, International Publication No. WO 01/59066 to Xencor, Inc., U.S. Pat. No. 6,278,794 to Parekh et al., and U.S. Patent Application No. 2001/0000807 to Freire and Luque.
Generation of structure-based pharmacophores, utilizing experimental methods such as X-ray crystallography or NMR, optionally in conjunction with protein structure determination methods, such as homology modeling, is known in the art. However, in order for this approach to be employed it must be possible to obtain appropriate data from the ligand in the conformation specific for the receptor defining the pharmacophore. In many, if not most, instances this is not feasible.
It may be determined that a particular peptide or protein sequence, with a length between about five residues to about fifty or more residues, binds to a particular receptor. However, the specific residues actually participating in binding, and the local secondary structure of the sequence which contains these specific residues, is not known. Without this knowledge, it is impossible to devise a systematic rational approach to make peptide-based drugs, peptidomimetic drugs or small molecule drugs. With knowledge of the specific residues and local secondary structure, it is possible to define the pharmacophore for the receptor. This definition may include, for example, the location in a three-dimensional construct of hydrogen bond donors and acceptors, positively and negatively charged centers, aromatic ring centers, hydrophobic centers and the like, such as described in terms of the distances between the atoms in the pharmacophore.
U.S. Pat. No. 5,834,250, to Wells et al., provides methods for the systematic analysis of the structure and function of polypeptides, specifically by identifying active domains by substituting a “scanning amino acid” for one of the amino acid residues within a suspected active domain of the parent polypeptide. These residue-substituted polypeptides are then assayed using a “target substance”. In practice, a “scanning amino acid”, such as alanine, is substituted for various residues in a polypeptide, and binding of the substituted polypeptide to a target substance compared to binding of the parent polypeptide. Similarly, U.S. Pat. No. 6,084,066, to Evans and Kini, discloses homologs and analogs of naturally occurring polypeptides with “conformation-constraining moieties” flanking “interaction sites”. However, this method requires that the “interaction site” or amino acid sequence be known. The “interaction site” sequence is then flanked on both termini with proline residues, which are asserted to stabilize interaction sites.
There is thus a significant and substantial need to develop methods for identifying the specific residues in a peptide which are involved in binding to a receptor of interest, and to identify the specific secondary structure of the residues involved in binding.
Metallopeptides. It is known that linear peptides have high rotational degrees of freedom, such that for even small peptides with known primary structures the theoretically possible secondary and tertiary structures may number in the millions. In general cyclic peptides are more constrained, and at least small cyclic peptides have far fewer theoretically possible secondary and tertiary structures. However, even with cyclic peptides it is frequently impossible to predict with precision the actual secondary structures present in such peptide. By contrast, metallopeptides have well-defined and limited secondary structures, with the residues involved in metal ion complexation forming a turn structure about the metal ion. The atoms forming a part of the coordination sphere of the metal ion are fixed by the coordination geometry of the metal ion. This, coupled with the peptide bonds between residues and the side chain bonds, yields a conformationally fixed and predictable secondary structure for at least the residues involved in metal ion complexation. U.S. Pat. No. 5,891,418, entitled Peptide-Metal Ion Pharmaceutical Constructs and Applications, U.S. Pat. No. 6,027,711, entitled Structurally Determined Metallo-Constructs and Applications, and P.C.T. Patent Application Serial No. PCT/US99/29743, Published Application No. WO 96/40293, entitled Metallopeptide Combinatorial Libraries and Applications, each teach aspects of making and using metallopeptides and mimetics thereof, and each of the foregoing is incorporated herein by reference. These patents and applications disclose receptor-specific metallopeptides and methods of making peptides and complexing the peptides to various metal ions.
There are methods for screening peptides for metal coordinating properties, such as disclosed in U.S. Pat. No. 6,083,758 to Imperiali and Walkup. However, these methods, which employ monitoring the fluorescence to detect metal coordination, do not provide any information regarding binding of metal coordinated peptides to receptors or targets of interest.
There is thus a need for identifying target-specific sequences within peptides and proteins, and is further a need for a knockout method, which by demonstrating decreased or changed binding or functionality of selected constructs elucidates the primary sequence involved in such binding or functionality.